1. TUBEX ® TF TUBEX ® TF HOW TO PERFORM THE ASSAY TO ENSURE CORRECT RESULTS IDL Biotech AB (publ) 2007

2. TUBEX ® TF Introduction Introduction Assay procedure Assay procedure Assay principle Assay principle Interpretation of results Interpretation of results Summary Summary TUBEX ® TF rapid typhoid detection

3. TUBEX ® TF INTRODUCTION TUBEX ® TF rapid typhoid detection

4. Typhoid Fever (TF) Facts Annual occurrence ~ 22 million cases Annual occurrence ~ 22 million cases – ~ 500.000 deaths worldwide Endemic disease, particularly in Asia, Africa and South America Endemic disease, particularly in Asia, Africa and South America Humans are the only natural host & reservoir Humans are the only natural host & reservoir –Causative organism Salmonella typhi Disease transmission by contaminated food and water Disease transmission by contaminated food and water WHO, Background document on typhoid fever, WHO/V&B/03.07 /update 2004 TUBEX ® TF rapid typhoid detection

5. Issues in TF Management Un- & under diagnosed disease Un- & under diagnosed disease Targeting of correct management Targeting of correct management Rural disease – lack of diagnostic prerequisites Rural disease – lack of diagnostic prerequisites Unnecessary high mortality rates Unnecessary high mortality rates Minimizing disease spread and development of multidrug resistance Minimizing disease spread and development of multidrug resistance TUBEX ® TF rapid typhoid detection

6. TUBEX ® TF Introduction Rapid and simple serum test Rapid and simple serum test –Less than 10 minutes Specific IgM detection (O9 antigen) Specific IgM detection (O9 antigen) –Only current infection No need for heavy instrumentation No need for heavy instrumentation –Analysis performed in specific test strips –Results obtained by magnetic separation TUBEX ® TF rapid typhoid detection

7. TUBEX ® TF Clinical Benefits TUBEX ® TF specifically detects IgM, thus: Immediate indication of acute TF Immediate indication of acute TF –One test gives direct result –No need for additional antibody class separation from the patient’s serum Enhanced diagnostic specificity Enhanced diagnostic specificity No false results due to previous infections and existing IgG No false results due to previous infections and existing IgG Tam et al, J Immunol Methods, 282;83, 2003 TUBEX ® TF rapid typhoid detection

8. TUBEX ® TF score frequency distribution in an endemic area typhoid patients (upper) typhoid patients (upper) control individuals (lower) control individuals (lower) N:o of samples TUBEX score Lim et al, J Clin Microbiol, 36:8;2271, 1998 TUBEX ® TF Endemic scores TUBEX ® TF rapid typhoid detection

9. TUBEX ® TF 1 st comparison Assay Sensitivity (%) Specificity (%) TUBEX ® TF 100100 ELISA IgM 10096.9 Widal (TO+TH) 81.343.3 Direct slide agglutination 18.291.3 Inhibition slide agglutination 38.5100 Lim et al, J Clin Microbiol, 36:8;2271, 1998 TUBEX ® TF rapid typhoid detection

10. TUBEX ® TF Clinical data TUBEX TF Overall figures TUBEX TF Overall figures Sensitivity78 – 94% Specificity80 – 94% PPV 95-98%NPV 60% TUBEX TF Culture positive TUBEX TF Culture positive Sensitivity87 – 90% Specificity76 – 85% PPV 77-86%NPV 84-88% Jaffery; Kawano; Olsen. Ref’erences on request TUBEX ® TF rapid typhoid detection

11. TUBEX ® TF Unique assay format Easy to perform Based on an advanced technology TUBEX ® TF rapid typhoid detection

12. TUBEX ® technology What is unique for ? What is unique for TUBEX ® ? Special designed reaction wells Special designed reaction wells Special designed magnetic stands Special designed magnetic stands Visual reading compared to a color scale for interpretation of results Visual reading compared to a color scale for interpretation of results Inhibition binding in solution Inhibition binding in solution A semi quantitative rapid assay A semi quantitative rapid assay TUBEX ® TF rapid typhoid detection

13. TUBEX ® TF - components Brown Reagent – –SuperParamagnetc Particles coated with LPS O9 antigen TUBEX ® TF rapid typhoid detection

14. TUBEX ® TF - components Blue Reagent – –Blue Dyed Latex Microspheres coated with monoclonal IgM specific for LPS O9 antigen TUBEX ® TF rapid typhoid detection

15. TUBEX ® TF - components Kit controls – – Negative control – score 0-2 – – Positive control – score 8-10 TUBEX ® TF rapid typhoid detection

16. TUBEX ® TF ASSAY PROCEDURE TUBEX ® TF rapid typhoid detection

17. Assay procedure, preparations Room-temperature reagents and samples Room-temperature reagents and samples Place the Reaction Wells Strip upright on the bench Place the Reaction Wells Strip upright on the bench TUBEX ® TF rapid typhoid detection

18. Assay procedure, Brown Reagent Vortex the Brown Reagent. Vortex the Brown Reagent. Add 45µl Brown Reagent to each well to be used. Add 45µl Brown Reagent to each well to be used. TUBEX ® TF rapid typhoid detection

19. Assay procedure, sample Add 45µl of patient sample, positive control and negative control to appropriate wells Add 45µl of patient sample, positive control and negative control to appropriate wells TUBEX ® TF rapid typhoid detection

20. Assay procedure, incubation Mix thoroughly by pipetting up & down 10 times. Mix thoroughly by pipetting up & down 10 times. Incubate Reaction Wells Strip on the bench for 2 minutes. Incubate Reaction Wells Strip on the bench for 2 minutes. WAIT  2min TUBEX ® TF rapid typhoid detection

21. Assay procedure, Blue Reagent Vortex the Blue Reagent. Vortex the Blue Reagent. Add 90µl Blue Reagent to each well. Add 90µl Blue Reagent to each well. TUBEX ® TF rapid typhoid detection

22. Assay procedure, incubation Cover Reaction Wells Strip with Sealing Tape Cover Reaction Wells Strip with Sealing Tape Hold across and tilt Reaction Strip 90° Hold across and tilt Reaction Strip 90° Shake rapidly for 2 minutes Shake rapidly for 2 minutes SHAKE  2min TUBEX ® TF rapid typhoid detection

23. Assay procedure, separation Place the Reaction Strip on the magnetic stand (Color Scale) Place the Reaction Strip on the magnetic stand (Color Scale) Wait 5 minutes Wait 5 minutes Read the results Read the results WAIT  5min TUBEX ® TF rapid typhoid detection

24. TUBEX ® TF Assay procedure TUBEX ® TF Brown Reagent (45µl). 1 Sample or TUBEX ® TF Control (45µl). Mix 10x. 2 Incubate 2 min. 3 TUBEX ® TF Blue Reagent (90µl). 4 Separate 5 min on TUBEX ® Color Scale. Read results. 6 Cover strip. Hold across and tilt 90°. Shake 2 min. 5

25. TUBEX ® TF ASSAY PRINCIPLE TUBEX ® TF rapid typhoid detection

26. Assay principle Understanding the assay principle is vital to avoid errors due to performance TUBEX ® TF rapid typhoid detection

27. Assay principle immunoreaction between the LPS O9 antigen of S. Typhi and specific IgM antibodies patient IgM inhibit binding between Blue Reagent and Brown Reagent inhibition is proportional to the concentration of specific O9 IgM antibodies in the sample TUBEX ® TF rapid typhoid detection

28. Positive patient sample IgM antibodies in the sample bind to antigen on the paramagnetic microspheres. TUBEX ® TF rapid typhoid detection

29. Positive patient sample Bound patient antibodies inhibit the binding of Blue Reagent TUBEX ® TF rapid typhoid detection

30. Positive patient sample Result Color is from bluish to intense blue Score is read 4 to 10

31. Negative patient sample In a negative sample there is no inhibition – the coated particles bind to each other TUBEX ® TF rapid typhoid detection

32. Negative patient sample Result Color is pink Score is read 0 - 2 TUBEX ® TF rapid typhoid detection

33. Negative test Negative test absence of IgM O9 specific antibodies in absence of IgM O9 specific antibodies in patients serum patients serum Positive test Positive test presence of IgM O9 specific antibodies in presence of IgM O9 specific antibodies in patients serum patients serum The specificity is dependent on the right proportions of reagents and sample The specificity is dependent on the right proportions of reagents and sample Assay outcome TUBEX ® TF rapid typhoid detection

34. What can go wrong? / Error sources Wrong proportions of reagents and sample Non-homogeneous particle suspension Insufficient mixing during incubation Pipetting error Insufficient mixing of reagents Too slow shaking during incubation

35. What can go wrong? / Error sources Wrong samples Disturbed reaction mixture after separation EDTA or Citrate plasmas Icteric or hemolysed samples Re-separation of a disturbed reaction mixture

36. Too much Brown Reagent - results in binding of both patient IgM and assay IgM Error sources, false negative

37. Too much Blue Reagent or too little Brown Reagent – will cause saturated Brown Reagent – Leaving unbound Blue Reagent in the supernatant Error sources, false positive

38. TUBEX ® TF INTERPRETATION of RESULTS TUBEX ® TF rapid typhoid detection

39. TUBEX ® TF Assay result TUBEX ® TF rapid typhoid detection

40. TUBEX® TF Assay result Compare the color in each reaction well to the color scale on the magnetic stand Compare the color in each reaction well to the color scale on the magnetic stand The bluer the color in the supernatant the higher the concentration of antibodies in the sample The bluer the color in the supernatant the higher the concentration of antibodies in the sample Interfering colors in patient samples unables interpretation of results Interfering colors in patient samples unables interpretation of results TUBEX ® TF rapid typhoid detection

41. Interpretation Score Interpretation Guide ≤ 2 NEGATIVE – Does NOT indicate current typhoid fever infection. TUBEX  TF Negative Control 3 Borderline, inconclusive score. Repeat analysis. If still inconclusive, repeat sampling at a later date. 4 WEAK POSITIVE – Indication of current typhoid fever infection. 6 - 10 POSITIVE – Strong indication of current typhoid fever infection. TUBEX  TF Positive Control TUBEX ® TF rapid typhoid detection

42. TUBEX ® TF SUMMARY TUBEX ® TF rapid typhoid detection

43. For correct results! Use only serum or heparinized plasma Use only serum or heparinized plasma Avoid icteric or hemolysed samples Avoid icteric or hemolysed samples Make sure that particles are resuspended Make sure that particles are resuspended Make sure that the volumes are correct Make sure that the volumes are correct Mix Brown reagent and sample properly Mix Brown reagent and sample properly Mix thorougly after addition of Blue reagent Mix thorougly after addition of Blue reagent Do not re-separate a disturbed reaction mixture Do not re-separate a disturbed reaction mixture TUBEX ® TF rapid typhoid detection

44. TUBEX ® TF rapid typhoid detection IDL Biotech AB (publ)